We have recently shown that blockage of RNA polymerase II is sufficient to cause the induction of p53 in mammalian cells and may be a common mechanism by which p53 is stabilized following various cellular stresses. The cellular proteins MDM2 and JNK are known to bind p53 and stimulate p53 degradation. Recent studies show that both the and C-terminal domains of p53 are required for efficient degradation of p53. We have obtained preliminary results suggesting that ser315 of p53 is important in the regulation of p53 stability. Firstly, phosphorylation of ser315 enhanced the degradation of p53 in vitro. Secondly, inhibition of the ser315 kinases Cdc2 and Cdk2 via inhibition of the CDK-activating kinase (CAK), caused a stabilization of p53 in cells. Thirdly, we have found a phosphatase, hCDC14, which binds p53 and specifically dephosphorylates the ser315 site of p53 in vitro. In this proposal, we will further explore the potential role of ser315 phosphorylation in the regulation of p53 degradation and how blockage of RNA polymerase II may attenuate ser315 phosphorylation. In specific aim number 1, we will utilize a cell-free extract system established in the lab to study the degradation of p53 in vitro. Recombinant wild-type p53 will be phosphorylated specifically on ser315 and its stability in cell extracts will be assessed. Preliminary results suggest that phosphorylation of ser315 enhances degradation in vitro. We will also assess the stability of recombinant p53 mutated at the 315 site, or containing deletions of the C- or N-terminal domains. In specific aim number 2, we will investigate the role of the Cdk- activating kinase (CAK), Cdk2 and Cdc2 in the regulation of p53 stability. CAK is a component of the RNA polymerase II holoenzyme, and as such, it may be a sensor for RNA polymerase II elongation. We hypothesize that under normal conditions CAK will activate Cdc2 and Cdk2 which in turn will phosphorylate p53 on ser315. Following inhibition of RNA polymerase II elongation, the activity of CAK is attenuated leading to increased stability of p53 by abrogation of ser315 phosphorylation of p53. We will use dominant negative CAK, Cdc2 and Cdk2 to specifically inhibit these enzymes in cells and study whether inhibition of these kinases causes an increased stability of p53. Finally, in specific aim number 3, we will explore the role of the novel human p53 phosphatase, hCDC14, in the regulation of p53 stability. Preliminary studies have shown that hCDC14 binds p53 and specifically dephosphorylates ser315 of p53. We will express wild-type or mutant CDC14 in MCF7 cells and study what effect it will have on basal and induced levels of p53.